CN103923885A - Infectious bursal disease virus Vero cell-adapted strain and application thereof - Google Patents
Infectious bursal disease virus Vero cell-adapted strain and application thereof Download PDFInfo
- Publication number
- CN103923885A CN103923885A CN201410152417.XA CN201410152417A CN103923885A CN 103923885 A CN103923885 A CN 103923885A CN 201410152417 A CN201410152417 A CN 201410152417A CN 103923885 A CN103923885 A CN 103923885A
- Authority
- CN
- China
- Prior art keywords
- virus
- vero cell
- strain
- adapted strain
- cloacal bursa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an infectious bursal disease virus Vero cell-adapted strain and belongs to the field of bioengineering. The related infectious bursal disease virus Vero cell-adapted strain is named Ck /Jiangsu/ NJ-23/2008 and has a collection No. of CGMCCNO.8852. The infectious bursal disease virus Vero cell-adapted strain which can efficiently multiply on serum-free cultured Vero cell is finally obtained through wild strain separation, chick embryo passage, Vero cell passage adaption; the infectious bursal disease virus Vero cell-adapted strain is subjected to continuous passage culture on the serum-free cultured Vero cell and TCID50 can be kept to be higher than 108.5/mL. Virus culture solution is inactivated and prepared into oil emulsion; after the prepared oil emulsion is used to immunize chicken, detection proves that the prepared oil emulsion has good immunogenicity. The infectious bursal disease virus (IBDV) strain and the production process thereof are simple, safe and efficient and suitable for industrial culture.
Description
Technical field
The present invention relates to bioengineering field, be specifically related to infections chicken cloacal bursa virus Vero cell adapted strain and application thereof.
Background technology
Infectious bursal disease (Infectious Bursal Disease, IBD), it is a kind of height contagious disease of the chicken that caused by infectious bursa of Fabricius virus (IBDV), betide the earliest the Gamboro area in U.S. Delaware state, the young chicken in 3 ~ 6 week age of main infection, virus is breeding rapidly in the lymphocyte of the fabricius bursa, and the bone-marrow-derived lymphocyte of the damage fabricius bursa, causes serious immunosuppression.This disease is imported China big area into and is broken out and continue popularly after 1980, has had a strong impact on the development of China's poultry husbandry, currently generally adopts vaccination to prevent for this disease.
At present, the IBDV vaccine of domestic production normally adopts chick embryo fibroblast to cultivate virus of proliferation.Chick embryo fibroblast needs to add a certain amount of fresh serum in culturing process, conventional foetal calf serum or calf serum, but serum composition brings very large difficulty in the process of purification and harvested cell product, and the quality difference of every batch of serum, thereby affect the stability of vaccine, in vaccine use procedure, also may bring out anaphylaxis simultaneously.
Along with the continuous progress of animal cell non-serum culture technique, for the application of the animal cell large-scale serum-free culture technology including Vero cell provides necessary technical support, serum-free suspension culture has become the general trend that the biotech drug including vaccine is produced.
Current, the virus titer of the IBDV vaccine of domestic production is on the low side, and immunoprotection efficiency is on the low side; In the mode of production, still use the mode of production of rolling bottle inoculation IBDV, volume of culture 0.5L left and right, cannot prepare on a large scale.
Summary of the invention
The object of this invention is to provide infections chicken cloacal bursa virus Vero cell adapted strain, can on Vero cell, stablize propagation, do not need to add serum in culturing process, safe, virus titer is high, and immunogenicity is strong.
Another object of the present invention is to provide the preparation method of the virus liquid of described infections chicken cloacal bursa virus Vero cell adapted strain.Adopt microcarrier suspension culture technology, be applicable to large scale culturing, the method is simple, safety, the virus liquid obtaining tire height, steady quality.
A further object of the present invention is to provide described infections chicken cloacal bursa virus Vero cell adapted strain in the application of preparing in bursal disease vaccine; adopt after vaccine inoculation prepared by this infections chicken cloacal bursa virus Vero cell adapted strain; antibody titer is high; extended period is long, can effectively protect inoculation animal.
Object of the present invention adopts following technical scheme to realize.
A kind of infections chicken cloacal bursa virus Vero cell adapted strain, is infections chicken cloacal bursa virus Ck/Jiangsu/NJ-23/2008 strain, and deposit number is CGMCC NO. 8852.
The present invention also provides described infections chicken cloacal bursa virus Vero cell adapted strain in the application of preparing in bursal disease vaccine.
The present invention also provides the virus liquid of described infections chicken cloacal bursa virus Vero cell adapted strain.
The preparation method of the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain of the present invention, comprising: on the Vero of serum-free culture cell, breed described infections chicken cloacal bursa virus Vero cell adapted strain, obtain virus liquid.
In the present invention, in described infections chicken cloacal bursa virus Vero cell adapted strain breeding, Vero cell is attached on microcarrier, cultivates at zooblast reactor inner suspension.
In the present invention, described Vero cell adopts IVT culture medium culturing to obtain.
In the present invention, in described breeding, cell maintenance medium is the mixture of lactalbumin hydrolysate and IVT substratum.
The present invention also provides a kind of vaccine composition, it is characterized in that activeconstituents is the described infections chicken cloacal bursa virus Vero cell adapted strain of deactivation.
The present invention also provides the preparation method of described vaccine composition, by the virus liquid deactivation of described infections chicken cloacal bursa virus Vero cell adapted strain, then mixes with adjuvant, emulsification obtains vaccine composition.
Beneficial effect:
Infections chicken cloacal bursa virus Vero cell adapted strain provided by the invention can be stablized propagation on Vero cell, does not need to use serum in culturing process, and safe, virus titer is high, and immunogenicity is strong.
The invention provides the preparation method of the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain, adopt microcarrier suspension culture technology, be applicable to large scale culturing, the method is simple, safety, efficient, the virus liquid obtaining tire height, steady quality, be applicable to industrial amplification culture.
Adopt after vaccine inoculation prepared by described infections chicken cloacal bursa virus Vero cell adapted strain, antibody titer is high, and the extended period is long, can effectively protect inoculation animal.
Brief description of the drawings
Fig. 1 shows the passage number impact that strain is tired on IBDV virus N J-23.
Fig. 2 is the Electronic Speculum figure that connects the front Vero cell of poison.
Fig. 3 has shown the Vero cell cultures propagation IBDV process sticking on microcarrier.
Embodiment
Acquisition and the characteristic measurement of embodiment 1 IBDV virus
Adopt the doubtful infectious bursal disease material separating from China Jiangsu Province morbidity chicken group, inoculation no-special pathogen (Specific Pathogen Free, SPF) chicken embryo isolated viral, after this strain is bred on SPF chicken embryo, in chick embryo fibroblast, carry out plaque purification, by purifying, the screening of different plaques, finally obtain virus N J strain.
Virus N J strain is carried out to following properties qualification:
(1) virus N J strain amplification: by aseptic PBS damping fluid dilution 10 for virus N J strain
3doubly, with chorioallantoic membrane approach inoculation 10 ~ 11 age in days SPF chicken embryos, inoculum size is 0.2mL/ embryo, 37 DEG C of cultivations, the allantoic fluid of results chicken embryo in 36 ~ 48h.
(2) EID of virus
50: the chick embryo allantoic liquid that step (1) is obtained carries out 10 times of gradient dilutions with aseptic PBS damping fluid, gets dilution 10
6~ 10
9allantoic fluid doubly, inoculation 10 ~ 11 age in days SPF chicken embryos, inoculum size is 0.2ml/ embryo, put 37 DEG C of cultivations, discard chicken embryo dead in 24h, cut open inspection and observe after 24h to dead chicken embryo and the still idiosome situation of strong chicken embryo of living of 168h in 168h, dwindle by having the hemorrhage and idiosome of head, neck, be judged to infection.Detected result is: in the chick embryo allantoic liquid that step (1) obtains, viral level is 10
7.5eID
50/ mL.
(3) virus-specific qualification: set up virus control group and neutralization group, every group is provided with 5 10 ~ 11 age in days SPF chicken embryos.The allantoic fluid of step (1) results is diluted to 10 with aseptic PBS
4.0eID
50/ mL, mixes with isopyknic anti-infectious bursal disease specific serum (being purchased from China Veterinery Drug Inspection Office), in room temperature and after 1h, with chorioallantoic membrane approach inoculation neutralization group chicken embryo, each egg inoculation 0.2mL; Virus control group chicken embryo, inoculation same amount virus N J strain.168h after inoculation, neutralization group chicken embryo is strong living all, in virus control chicken embryo dead 4.All chick embryo allantoic liquids are carried out to chicken red blood cell aggegation experiment, all negative, illustrate that this virus N J strain is IBDV purified virus.
(4) viral pure property qualification: carry out bacterium, mould, mycoplasma and exogenous virus according to existing " Chinese veterinary pharmacopoeia " annex and detect, result is all negative.
(5) structural protein gene (VP2 gene) sequence of mensuration virus N J strain, its sequence is as shown in SEQ ID NO:1.The homology of virus N J strain VP2 gene order and current I BDV OKYM Japan highly virulent strain, the poisoning virulence vaccine strain of B87, PBG98 low virulent strain is all more than 90%, illustrate that virus N J strain is infections chicken cloacal bursa virus, called after infections chicken cloacal bursa virus NJ strain, is abbreviated as IBDV virus N J strain.
The screening of embodiment 2 infections chicken cloacal bursa virus Vero cell adapted strains
This enforcement is bred infections chicken cloacal bursa virus NJ strain on the Vero of serum-free culture cell, and by limiting dilution assay, screening obtains the IBDV virus N J-23 that a strain virus titre is high, have genetic stability characteristic.
Concrete grammar:
(1) Vero cell adopt IVT substratum (serum free medium is purchased from Gibico company) be cultured to 95% degree of converging, Vero cell is adopted to 0.1% trysinization after 1000rpm centrifugal, with fresh IVT substratum re-suspended cell.Cell is counted, according to 8 × 10
5paved 96 orifice plates in cell/ hole.
(2) screening: with to be added with final concentration be 0.25 ~ 2.5%(mass percentage concentration) the IVT substratum dilution NJ strain virus liquid of lactalbumin hydrolysate (Sigma), with 5 extent of dilution (10
-4～10
-8) virus liquid infect step (1) and be paved with the individual layer Vero cell of 96 orifice plates, put 37 DEG C and cultivate 120h, results virus liquid.Multigelation 3 times detects every hole virus titer TCID in chick embryo fibroblast
50(virus titer is measured detailed step with reference to " Chinese veterinary pharmacopoeia ").Culture higher virus titer is passaged to the individual layer Vero cell continuation cultivation that is paved with 24 orifice plates, observation of cell pathology (CPE) under microscope, gathers in the crops virus liquid after cultivation 120h.Repeated cloning purifying 3 times in the same way, screening obtains the high infections chicken cloacal bursa virus Ck/Jiangsu/NJ-23/2008 strain of virus titer, is abbreviated as infections chicken cloacal bursa virus NJ-23 strain.
(3) virus-specific qualification: set up virus control group and neutralization group, every group is provided with 5 10 ~ 11 age in days SPF chicken embryos.Infections chicken cloacal bursa virus NJ-23 strain nutrient solution is diluted to 10 with aseptic PBS
4.0tID
50/ mL, mixes with isopyknic anti-infectious bursal disease specific serum (being purchased from China Veterinery Drug Inspection Office), in room temperature and after 1h, with chorioallantoic membrane approach inoculation neutralization group chicken embryo, each egg inoculation 0.2mL; Virus control group chicken embryo, inoculation same amount virus N J-23 strain.Result shows: 168h after inoculation, neutralization group chicken embryo is strong living all, in virus control chicken embryo dead 4.All chick embryo allantoic liquids are carried out to chicken red blood cell aggegation experiment, all negative, illustrate that this virus N J-23 strain is IBDV purified virus.
(4) virus stability detects: IBDV virus N J-23 strain is gone down to posterity on Vero cell (adopt IVT culture medium culturing obtain), and detect the virus titer of each generation, the results are shown in Figure 1, find after 30 generations, still to keep higher virus titer being passaged to.
The preservation information of IBDV virus N J-23 strain is as follows:
Biomaterial (strain): Ck/Jiangsu/NJ-23/2008;
Classification And Nomenclature: infections chicken cloacal bursa virus;
Latin name: Infectious Bursal Disease Virus, (IBDV);
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC);
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica;
Preservation date: on March 6th, 2014;
Deposit number: CGMCC No. 8852.
Embodiment 3 cultivates IBDV virus N J-23 strain virus liquid on the Vero of serum-free microcarrier suspension culture cell
The method of cultivating IBDV virus N J-23 strain virus liquid on the Vero of serum-free microcarrier suspension culture cell is as follows:
(1) serum-free culture Vero cell: Cultivation of Vero in IVT substratum, when cell reaches 95% degree of converging, after the trysinization of employing 0.1%, 1000rpm is centrifugal, with fresh IVT substratum re-suspended cell, cell is divided according to a certain percentage and reached in new culturing bottle, continue to be cultured to cell and reach 95% degree of converging, obtain Vero cell.
(2) initial Vero cell is in serum-free microcarrier suspension culture: in IVT substratum, adding final concentration is 0.25%(mass percentage concentration) lactalbumin hydrolysate after obtain viral maintenance medium, its pH value is 7.1.0.1% trysinization for the Vero cell that step (1) is obtained, centrifugal after, be resuspended in fresh IVT substratum, after the centrifugal 10min of 1500rpm, get precipitation, acquisition Vero cell.By Vero cell according to 3 ~ 4 × 10
5the initial density of cell/mL adds microcarrier Cytodex 1(to be purchased from GE Healthcare company) in, add viral maintenance medium simultaneously, enter zooblast bioreactor culture.Culture condition: controlling medium pH value is 7.1,37 DEG C of suspension culture stirring velocity 35rpm, dissolved oxygen 30%, temperature.
(3) propagation of IBDV virus
:treat that the slimeball rate of Vero cell on microcarrier reaches 40 ~ 50%(Fig. 2), be 0.01 ~ 0.2 inoculation IBDV virus N J-23 strain according to infection multiplicity, virus of proliferation NJ-23 strain.In breeding, control 37 DEG C of medium pH values 7.2, suspension culture stirring velocity 35rpm, dissolved oxygen 30%, temperature.After connecing poison, 24h sampling is observed, and finds that Vero cell is still in growth phase, and Vero cell density can reach 5 ~ 8 × 10
5cell/mL; Meet the rear 48h of poison, part Vero cell comes off from microcarrier; Meet the rear 72h of poison, have 50% left and right cell detachment; Meet the rear 120h of poison, most of Vero cell comes off (Fig. 3) from microcarrier, results viral cultures.
(4) mensuration that virus liquid is tired: by viral cultures multigelation 3 times, releasing virus, obtains virus liquid.In the chick embryo fibroblast preparing, measure tiring of virus liquid.
By the same way, on the Vero of serum-free microcarrier suspension culture cell, cultivate infections chicken cloacal bursa virus conventional vaccine strain B87(and be purchased from China Veterinery Drug Inspection Office); Adopt ordinary method in chick embryo fibroblast, to cultivate infections chicken cloacal bursa virus NJ strain.
Result shows:
IBDV virus N J-23 strain is cultivated in the Vero of serum-free microcarrier suspension culture cell proliferation, at the TCID that tires of 24h virus liquid
50be 10
4.5/ mL, at the TCID that tires of 120h virus liquid
50be 10
8.5/ mL.
Infections chicken cloacal bursa virus conventional vaccine strain B87 breeds on the Vero of serum-free microcarrier suspension culture cell, at the TCID that tires of 120h virus liquid
50for 10
6.0/ mL;
Infections chicken cloacal bursa virus NJ strain is cultivated in chick embryo fibroblast, the high-titer TCID of virus liquid
50be 10
7.0/ mL.
Result shows, the TCID of the IBDV virus N J-23 strain by serum-free microcarrier suspension culture Vero cell proliferation
50far above conventional vaccine strain B87, simultaneously also high than the virus titer of conventional chick embryo fibroblast propagation IBDV NJ strain.
Through many experiments, find IBDV virus N J-23 strain continuous passage cultivation on the Vero of serum-free culture cell, TCID
50for remaining on 10
8.5more than/mL.
The immune effect of embodiment 4 IBDV virus N J-23 strain vaccines
In order to verify the immunogenicity of IBDV virus N J-23 strain, make immunity after vaccine, detect antibody titer, extended period, attack malicious Protection; The immune commercial seedling with based article simultaneously: infectious bursal disease inactivated vaccine (G strain, Harbin Wei Ke biotechnology development company), relatively virus immunity originality.
(1), virus liquid and vaccine preparation: prepare IBDV virus N J-23 strain virus liquid according to embodiment 3 methods, connect the rear 120h of the cultivation results of poison virus liquid.Virus liquid is TCID after testing
50tire and be more than or equal to 10
8.0eID
50after/mL, obtain inactivation of viruses liquid through formalin-inactivated.Be that 96:4 mix with tween-80 according to volume ratio by inactivation of viruses liquid, obtain water.Be that 94:6 mix acquisition oil phase with Si Ben-80 according to volume ratio by white oil.Be that 1:3 mix with water according to volume ratio by oil phase, obtain IBDV virus N J-23 strain vaccine.
(2), SPF chicken: SPF chicken embryo, purchased from purchased from poultry Jinan, Shandong Sai Si poultry Science and Technology Ltd., through hatching to going out shell voluntarily, is raised in shield retaining.
(3), experimental animal grouping and immunity
Get 70 of 21 age in days SPF chickens, select at random 30 as NJ-23 strain immune group, every subcutaneous inoculation IBDV virus N J-23 strain vaccine 0.2mL; Select 30 else as conventional vaccine immune group, every subcutaneous inoculation is with commercial seedling (G strain, the Harbin Wei Ke biotechnology development company) 0.2mL of based article; Remaining 10, the not immune blank group of doing.After immune vaccine 1,2,3,4,5,6,7,8,9,10,11,12,13,14W(week), blood sampling separation of serum, measures infections chicken cloacal bursa virus neutralizing antibody.
(4), antibody titer detects
Adopt fixed virus diluted blood heat-clearing method to carry out serum neutralization test (neutralization experiment detailed step is with reference to " Chinese veterinary pharmacopoeia "), detect infections chicken cloacal bursa virus antibody titer in serum.When after 2500 times of serum dilutions still can in and 100 TCID
50virus, do not produce pathology, think qualified, be judged to positive serum.
(5), attack poison and the detection of protection efficiency
4W after immunity, attacks poison from 10 chickens of immune group (NJ-23 strain immune group and conventional vaccine immune group) random choose, 5 chickens of blank group.Other 5 chickens of blank group are as negative control group, the not immune poison of not attacking.Specifically attack malicious method as follows: will attack poison strain standard virulent strain BC6-85(and be purchased from Chinese medicine inspecting institute) be diluted to 10
5bID/ml(chicken body fabricius bursa infective dose), per oral inoculation, 200uL/ is only.Inoculate latter 3 days and cut open inspection, observe pathology.
(6), result is described
After 6.1 immunity, antibody horizontal detects: the 1. chicken of NJ-23 strain immune group, 2W after immunity, antibody positive rate is 20%, 3,4W antibody positive rate is elevated to 83.3%, 96.7%, illustrates that the IBDV virus N J-23 strain immunogenicity of being bred by serum-free microcarrier suspension is fine.After immunity, 4W antibody titer reaches the highest, is maintained to after 12W, and antibody positive rate starts to decline.2. conventional vaccine immune group, 2W after immunity, fails to detect antibody, and after immunity, 4W antibody positive rate is the highest, is increased to 83.3%, is maintained to after 10W, and antibody positive rate starts to decline.(table 2)
4W after 6.2 immunity, attack malicious result as shown in table 1: the 1. chicken of NJ-23 strain immune group, to attack poison with BC6-85 and within latter 3 days, cut open inspection and show that the fabricius bursa is without obvious pathology, chest muscle, leg flesh are normal; 2. the chicken of conventional vaccine immune group, attacks poison with BC6-85 and within latter 3 days, cuts open inspection and show, in 10 chickens, occur 1 fabricius bursa chest muscle hemorrhage, 1 there is fabricius bursa atrophy phenomenon; 3. blank group is cutd open the demonstration of inspection result, and different pathologies all appears in 5 chickens, and the fabricius bursa has damage in various degree, has also occurred chest muscle and leg flesh bleeding simultaneously, and this is that the typical fabricius bursa infects illness; 4. negative control group is without obvious virus.The above results explanation, compares and conventional vaccine group, and NJ-23 strain vaccine can more effectively be protected the attack of immune chicken opposing BC6-85 virus.
Table 1 challenge test result
Antibody positive rate after table 2 NJ-23 oil seepage and conventional seedling immunity
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> infections chicken cloacal bursa virus Vero cell adapted strain and application thereof
<130> 201404161
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1366
<212> DNA
<213> infections chicken cloacal bursa virus Ck/Jiangsu/NJ-23/2008 strain
<400> 1
atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60
ccaacaaccg gaccggcgtc cattccggac gacaccctgg agaagcacac tctcaggtca 120
gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
cctggattcc cgggctcaat tgtaggtgct cactacacac tgcaaggcaa tgggaactac 240
aagttcgatc agatgctcct gactgcccag aacctaccgg ccagttacaa ctactgcagg 300
ctagtgagtc ggagtctcac agtgaggtca agcacacttc ctggtggcgt ttatgcacta 360
aacggcacca taaacgccgt gaccttccaa ggaagcctga gtgaactgac agatgttagc 420
tacaatgggt tgatgtctgc aacagccaac atcaacgaca aaatcgggaa cgtcctagta 480
ggggaagggg tcaccgtcct cagcttaccc acatcatatg atcttgggta tgtgagactt 540
ggtgacccca ttcccgcaat agggcttgac ccaaaaatgg tagccacatg tgacagcagt 600
gacaggccca gagtctacac cataactgca gccgatgatt accaattctc atcacagtac 660
caaccaggtg gggtaacaat cacactgttc tcagctaata tcgatgccat cacaagcctc 720
agcgttgggg gagagctcgt gtttcaaaca agcgtccacg gccttgtact gggcgccacc 780
atctacctca taggctttga tgggacaacg gtaatcacca gggctgtggc cgcaaacaat 840
gggctgacga ccggcaccga caaccttatg ccattcaatc ttgtgattcc aacaaacgag 900
ataacccagc caatcacatc catcaaactg gagatagtga cctccaaaag tggtggtcag 960
gcaggggatc agatgtcatg gtcggcaaga gggagcctag cagtgacgat ccatggtggc 1020
aactatccag gggccctccg tcccgtcacg ctagtggcct acgaaagagt ggcaacagga 1080
tccgtcgtta cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca 1140
aagaacctgg ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg 1200
atactgagtg agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact 1260
gactttcgtg aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga 1320
gcattcggct tcaaagacat aatccgggcc ataaggagga tagctg 1366
Claims (9)
1. an infections chicken cloacal bursa virus Vero cell adapted strain, is characterized in that described Vero cell adapted strain is infections chicken cloacal bursa virus Ck/Jiangsu/NJ-23/2008 strain, and deposit number is CGMCC NO. 8852.
Described in claim 1 infections chicken cloacal bursa virus Vero cell adapted strain in the application of preparing in bursal disease vaccine.
3. the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain described in claim 1.
4. the preparation method of the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain described in claim 3, is characterized in that: on the Vero of serum-free culture cell, breed described infections chicken cloacal bursa virus Vero cell adapted strain, obtain virus liquid.
5. the preparation method of the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain according to claim 4, is characterized in that: in described breeding, Vero cell is attached on microcarrier, cultivates at zooblast reactor inner suspension.
6. according to the preparation method of the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain described in claim 4 or 5, it is characterized in that: described Vero cell adopts IVT culture medium culturing to obtain.
7. the preparation method of the virus liquid of infections chicken cloacal bursa virus Vero cell adapted strain according to claim 6, is characterized in that: in described breeding, cell maintenance medium is the mixture of lactalbumin hydrolysate and IVT substratum.
8. a vaccine composition, is characterized in that activeconstituents is infections chicken cloacal bursa virus Vero cell adapted strain described in the claim 1 of deactivation.
9. the preparation method of vaccine composition described in claim 8, is characterized in that the virus liquid deactivation of infections chicken cloacal bursa virus Vero cell adapted strain described in claim 3, then mixes with adjuvant, emulsification obtains vaccine composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410152417.XA CN103923885B (en) | 2014-04-16 | 2014-04-16 | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410152417.XA CN103923885B (en) | 2014-04-16 | 2014-04-16 | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103923885A true CN103923885A (en) | 2014-07-16 |
| CN103923885B CN103923885B (en) | 2016-05-04 |
Family
ID=51142284
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410152417.XA Expired - Fee Related CN103923885B (en) | 2014-04-16 | 2014-04-16 | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103923885B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368794A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Method of utilizing stirred bioreactor to produce infectious Bursal disease virus |
| CN106282098A (en) * | 2016-08-31 | 2017-01-04 | 天津瑞普生物技术股份有限公司 | A kind of utilize relatively low serum content nutrient cultivate DF1 cell with the method preparing infections chicken cloacal bursa virus |
| CN110272864A (en) * | 2019-07-25 | 2019-09-24 | 北京鼎持生物技术有限公司 | A kind of Vero33 cell strain adapting to serum free suspension culture and its acclimation method and application |
| CN112094819A (en) * | 2020-08-13 | 2020-12-18 | 浙江美保龙生物技术有限公司 | Full suspension culture method of chicken infectious bursal disease virus |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919461A (en) * | 1989-10-18 | 1999-07-06 | Akzo Nobel N.V. | Nonpathogenic infectious bursal disease virus and vaccine |
| CN102268411A (en) * | 2011-08-15 | 2011-12-07 | 江苏省农业科学院 | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation |
| CN103263667A (en) * | 2013-05-09 | 2013-08-28 | 北京中海生物科技有限公司 | Chicken infectious bursal disease live vaccine and production method thereof |
-
2014
- 2014-04-16 CN CN201410152417.XA patent/CN103923885B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919461A (en) * | 1989-10-18 | 1999-07-06 | Akzo Nobel N.V. | Nonpathogenic infectious bursal disease virus and vaccine |
| CN102268411A (en) * | 2011-08-15 | 2011-12-07 | 江苏省农业科学院 | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation |
| CN103263667A (en) * | 2013-05-09 | 2013-08-28 | 北京中海生物科技有限公司 | Chicken infectious bursal disease live vaccine and production method thereof |
Non-Patent Citations (4)
| Title |
|---|
| MUHAMMAD HIDAYAT RASOOL等: "Preparation and evaluation of Vero-cell infectious bursal disease vaccine in Pakistan", 《VACCINE》 * |
| 李健强 等: "传染性法氏囊病Vero细胞灭活疫苗的研究", 《西北农业大学学报》 * |
| 石岗 等: "传染性法氏囊病病毒Vero细胞培养条件优化的研究", 《中国兽医杂志》 * |
| 袁维峰 等: "三株鸡传染性法氏囊病毒弱毒株的分离与分子鉴定", 《中国动物传染病学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368794A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Method of utilizing stirred bioreactor to produce infectious Bursal disease virus |
| CN106282098A (en) * | 2016-08-31 | 2017-01-04 | 天津瑞普生物技术股份有限公司 | A kind of utilize relatively low serum content nutrient cultivate DF1 cell with the method preparing infections chicken cloacal bursa virus |
| CN106282098B (en) * | 2016-08-31 | 2019-10-29 | 天津瑞普生物技术股份有限公司 | A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus |
| CN110272864A (en) * | 2019-07-25 | 2019-09-24 | 北京鼎持生物技术有限公司 | A kind of Vero33 cell strain adapting to serum free suspension culture and its acclimation method and application |
| CN112094819A (en) * | 2020-08-13 | 2020-12-18 | 浙江美保龙生物技术有限公司 | Full suspension culture method of chicken infectious bursal disease virus |
| CN112094819B (en) * | 2020-08-13 | 2022-10-14 | 浙江美保龙生物技术有限公司 | Full suspension culture method of chicken infectious bursal disease virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103923885B (en) | 2016-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111632137B (en) | Triple vaccine for feline calicivirus disease, feline infectious rhinotracheitis and feline panleukopenia as well as preparation method and application thereof | |
| CN102258777B (en) | Method for preparing inactivated vaccine and combined vaccine by breeding infectious bursal disease virus (IBDV) by chicken embryo source cell line | |
| CN102260649B (en) | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor | |
| CN102220287B (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN111041002B (en) | Bivalent inactivated vaccine of porcine epidemic diarrhea virus variant 2a and 2b and preparation method thereof | |
| CN103923885B (en) | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof | |
| CN104195116B (en) | A kind of recombinant Newcastle disease virus and its construction method for expressing goose parvovirus VP3 genes | |
| CN102727884B (en) | Combined live vaccine against porcine reproductive and respiratory syndrome and pseudorabies, and preparation method thereof | |
| CN102166352B (en) | Preparation method of swine influenza bivalent inactivated vaccine and product of swine influenza bivalent inactivated vaccine | |
| CN104338127A (en) | Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine | |
| CN104587460A (en) | Mink viral enteritis and canine distemper binary living vaccine as well as preparation method and application thereof | |
| CN102488893B (en) | Duck hemorrhagic oophoritis inactivated vaccine production method by using cell line and product thereof | |
| CN102268411A (en) | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation | |
| CN102727883B (en) | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof | |
| CN103382460B (en) | Strain of Marek's disease virus vaccine strain, and separation and identification and application thereof | |
| CN103937753B (en) | H9N2 subtype avian influenza virus strain and inactivated vaccine thereof and application | |
| CN109207436B (en) | Group I type 4 avian adenovirus strain and application thereof | |
| CN102727877A (en) | Method for preparing highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) by utilizing bioreactor and application thereof | |
| CN102805862B (en) | Preparation method for SFTS bunyavirus purification and inactivation vaccines through VERO cell culture | |
| CN108315306A (en) | One plant height fertility swine fever virus and its construction method | |
| CN104073470A (en) | Spinner-flask culture method for H9N2 subtype of avian influenza virus | |
| CN102965345B (en) | Porcine parvovirus, vaccine composition and application thereof | |
| CN104164408A (en) | Newcastle disease, infectious bronchitis and avian influenza resisting vaccine composition and preparation | |
| CN103160475B (en) | Enterovirus 71 type viral strain, its application, vaccine and preparation method | |
| CN104195114A (en) | Avian pneumovirus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160504 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |